In this renewal proposal, we continue our work demonstrating the critical role of the Rho GTPase, Cdc42, in the molecular control of endothelial cell (EC) lumenogenesis in early blood vessel assembly during vasculogenesis. Our overall hypothesis is that Cdc42 represents a central signaling molecule in EC lumen formation and tube morphogenesis because of its key role in cell polarity pathways as well as coordinating cell signaling pathways controlling vesicular trafficking and fusion events involving intracellular vacuoles that are fundamental to this process. During this last funding period, we have made essential progress in demonstrating the function of Cdc42 in lumen formation as well as the identification of critical downstream Cdc42 effectors required for these events. Also, we have shown that intracellular vacuole formation and fusion are critical regulators of lumenogenesis both in vitro and in vivo during Zebrafish development (collaborative work with Dr. Brant Weinstein). We are able to label and visualize the intracellular vacuoles in vitro and in vivo using GFP-Cdc42 which further suggests a functional role for Cdc42 during these events. Also, we have developed essential experimental approaches such siRNA technology and its functional application in our morphogenic systems, real-time image analysis of EC lumenogenesis in 3D collagen matrices, proteomic capture techniques during tube morphogenesis to identify Cdc42-associated proteins which regulate lumen formation and identification of critical signal transduction cascades that control these events. We propose three specific aims which are;Specific Aim #1: To determine the molecular mechanisms by which the Cdc42 effectors, Pak2 and Pak4, control EC lumen and network formation in 3D collagen matrices. Specific Aim #2: To define how cell polarity signaling pathways involving Cdc42, Par3, Par6 and PKC zeta control the EC lumen formation mechanism in 3D collagen matrices. Specific Aim #3: To investigate the role of JamC and JamB in EC lumen formation and identify and characterize upstream Cdc42 GEFs which activate Cdc42 to control these events. PUBLIC HEALTH RELEVANCE: This work focuses on the ability of blood vessel lining cells to tube-like structures within three- dimensional environments such as collagen, which is a major structural component of tissue. These lining cells carry a group of proteins that control their ability to assemble into a cell-lined tube and which are required to assemble cellular networks of vessels to develop our blood vascular system. A basic understanding of the mechanisms underlying how blood vessels form is critical in efforts to stimulate or inhibit the process in the context of various human diseases such as cardiovascular disease or cancer.